Disposable electrophoresis unit

ABSTRACT

This invention relates to an expendable electrophoresis test unit for particle separation in a buffered system using direct current. The buffer is in semi-solid form which can be prepackaged with the unit, or dispensed from a tube at the time of use. The semi-solid buffer is prepared by mixing a liquid buffer solution with a thickening agent, preferably fumed silica. The unit itself is made of inexpensive disposable materials.

United States Patent Tocci 1 1 Feb. 6, 1973 [541 DISPOSABLEELECTROPHORESIS 3,432,414 3/1969 Rand ..204/180 G UNIT 3,494,846 2/1970Arquembourg ..204/180 G [75] inventor: Paul M. Toccl, Miami, Fla,Primary Examiner john H Mack [73] Assignee: TLC Corporation, Miami, Fla.Assistant Examiner--A. C. Prescott [22] Filed Sept 2 1971Att0rneyMeredith P. Sparks Appl. No.: 177,284

US. Cl ..204/180 G, 204/180 S, 204/299 Int. Cl. ..B0lk 5/00 Field ofSearch ....204/180 G, 180 R, 180 S, 299

References Cited UNITED STATES PATENTS Paglia et a1. ..204/299 Oliva 6!a1. ..204/299 [57] ABSTRACT This invention relates to an expendableelectrophoresis test unit for particle separation in a buffered systemusing direct current. The buffer is in semi-solid form which can beprepackaged with the unit, or dispensed from a tube at the time of use.The semi-solid buffer is prepared by mixing a liquid buffer solutionwith a thickening agent, preferably fumed silica. The unit itself ismade of inexpensive disposable materials.

10 Claims, 6 Drawing Figures PATENTED FEB 6 I975 I [III/III l INVENTORP/W/L M 7066/ BY 14 TTORNE X DISPOSABLE ELECTROPHORESIS UNIT Thisinvention relates to the separation of mixtures by differentialmigration of components through a transport medium in an electric field,and more particularly to an expendable electrophoresis test unit forparticle separation in a buffered system to which direct current isapplied.

The separation and consequent grouping of materials by electrophoresisoccurs within a buffered liquid support medium, wherein those particlesof a test sample that have a greater charge will migrate at a fasterrate and further in a given time than those of a lesser charge. Afterparticle grouping, identification of the materials in the test sample isdetermined by means known in the art.

The technique of electrophoresis involves the use of electricity,specifically, a constant potential difference or a constant amperageacross a medium to move compounds according to their electric charge.Many media have been used as the support, e.g., paper, agar-gel,cellulose acetate strips, polyacidamide gels, starch blocks, thin layerplates and others. Whatever the media, buffers are used as electrolytesat each end, the anode end and the cathode end. The buffers may beformulated according to many formulas and are tailored to particularseparations in particular media of certain specific groups of compoundsby varying not only their pH but their tonicity. These formulations arewell known in the field and are used extensively.

One of the drawbacks of electrophoresis has been the use of thesebuffers as liquids and the location in the many different shapes ofchambers now on the market. The liquid buffers are subject toevaporation with resultant loss of effectiveness due to pH changes andchanges in tonicity due to increased salt production. It is notdifficult to change a buffer gone bad, but it is a bothersometime-consuming chore. Also when one wishes to analyze a different typeof compound one must change buffers and clean the tank, wicks andchamber dividers. During the course of electrophoresis heat is evolvedand many elaborate schemes have been devised to maintain constanttemperature to keep radial diffusion to a minimum.

The buffer solutions are probably the greatest variable in the techniqueof electrophoresis. The problems of technique when liquid solutions areused as buffers are such that fairly elaborate mechanical systems areordinarily required. ln case of a gel media, for example, the directionof travel of the current necessitates a wick from the liquid buffersolution to the gel.

1 have now discovered that most of the objections to the use ofelectrophoresis with liquid buffer solutions can be overcome by the useof pre-mixed, semi-solid buffers dispensed from a closed container suchas a plastic or metal tube. The semi-solid buffer solution is dispenseddirectly into the chamber or well of the electrophesis tank so that thehands need not touch the buffer. Also, only as much material as isneeded is put into the chamber so that there is much less waste ofexpensive reagents. If the containers are relatively airtight andlight-tight, they can be stored in a refrigerator or freezer withoutextensive degradation for extended time periods. The containers can besmall and many can be stored in a very small area. Large batches ofmixtures can be made at one time and stored. With uniform buffersavailable, results obtained at different times and in different placeswill be more comparable than is now possible. The use of inexpensive,expendable, self-contained electrophoresis equipment will enable therelatively small clinic to use diagnostic procedures which heretoforehave been fairly well restricted to use by large commercial andinstitutional laboratories.

In a preferred embodiment of my invention, the buffer solution issuspended in particles of fumed silica. When the proportions of thefumed silica and buffer solution are correct the buffer in semi-solidform is then put into dispensers which may be of the syringe type or thecollapsible tube type. As a general indication of the approximatethickness, an amount of themixed buffer solution is mixed with an amountof a thickening agent such that the beaker or other vessel in whichmixing takes place can be turned upside down without the semi-solidproduct spilling out. The proportions of the ingredients will varywidely with the type of buffer mixtures employed. Generally, the amountof thickening agent varies from about 1 to about l5 percent by weight ofthe buffer solution used. Acid and alkaline emulsifiers, solidifiers,thixotropic agents, gelling agents and dispersing agents may be usedalso to form the semi-solid or to improve the characteristics of aparticular mixture. In this way volatile chemicals which are used asbuffers may be stored for many months so as to be ready forinstantaneous use.

According to my invention, the buffer solution in semi-solid form isplaced in each of a pair of spaced wells in a tank of light weightdisposable material, a

support medium wetted with the buffer is positioned at the top of thetank, electrodes for connecting to a power source for direct current areprovided at each well in electrical contact with the buffer contained inthe wells in order to provide passage of current through the semi-solidbuffer placed in the wells and at the top of the tank to cause chargedparticle migration for particle group identification.

The container is preferably made of plastic, for example, Mylar. The lidand body of the container may be made of the same or different type ofplastic. For example, the lid may be made of a clear plastic such as anacrylic plastic, with the base portion of the container made of a sturdypolystyrene. In the latter instance the inexpensive disposable tankhaving a pair of integrally formed spaced wells at each end isprepackaged with the buffer in semi-solid form placed in the wells andthe unit is covered by a thin sheet made preferably of metal foil orplastic to act as a seal unit the tank is ready for use.

A support medium, comprising for example, paper, a cellulose acetatestrip, a thin layer plate on a plastic backing, or agar gel ispositioned at the top of the tank. A buffer can be spread on the supportmedium, at the time of use, for example, by means of-a wiper, or byblotting with a towelette. The sample to be separated or identified isthen applied to the support medium which is in contact with thesemi-solid buffer in each end of the well. The lid is then fitted ontothe tank.

The buffer in semi-solid form can be stored in a separate container foruse when needed. Alternately, the chamber can be prepackaged with thesemi-solid buffer already placed in the wells of the tank.

In the latter instance the invention, comprising an inexpensivedisposable tank having a pair of integrally formed spaced wells at eachend, is prepackaged with buffers in semi-solid form, which buffers arecovered by a thin sheet made preferably of metal foil or a thin plasticsheet to act as a seal until the tank is ready for use.

The top for the tank may be inert plastic or may consist of a supportmedium, for example, of paper, a cellulose acetate strip, or a thinlayer plate on a plastic backing, on which is spread at the time of usean appropriate buffer to wet the medium. This can be done, for example,by means of a wiper, or by blotting with a towelette. Alternatively, awet support media may be used such as agar-gel or polyacidamide gel. Thesample to be separated or identified is then applied to the medium and alid fitted onto the tank. The container may be cooled by placing in aseparate specially designed container, or by other means.

In a preferred embodiment of my invention the top snaps onto a ridgearound the tank, such that each end of the support media is in contactwith the semi-solid buffer in each end of the well. The tank may vary insize and shape. When the tank is rectangular in shape the lid which actsas a closure may be hinged to the tank along one of the edges and araised rib along the remaining edges adapted frictionally to engage thelid along its unhinged edges to close the tank to the atmosphere.

An electrode of suitable electrical conducting properties is providedfor retention within each of the buffer wells as a foil. The foil isshaped to substantially fit within the wells. Each foil electrode mayhave an outwardly extending tab portion which is adapted to protrudefrom the well. The tabs or the electrodes are connected to a suitablesource of direct current, preferably through a switch.

In another embodiment of my invention the power supply is connected totwo electrodes which are adapted to pierce the buffer chambers throughpredetermined holes in the cover of each well. An electrode may be usedon each side of the top in the shape of a long knife-edge, each wedgebeing connected to the power source. Electrodes such as nichrome orplatinum wire may also be used. The electrodes may also be painted orprinted in the buffer well.

Any power supply which can deliver from to 5,000 volts and from one to100 milliamperes of direct current may be used. A special adaptor forthe electrodes may be necessary for each different size of tank used.

In order to electrophoretically separate materials by differentialmigration in an electrically conductive buffer system subjected to flowof current therethrough utilizing the disposable equipment of thisinvention, the test sample, which is subject to electrophoretic particleseparation, is deposited on the buffered support media. The semi-solidbuffer may be placed in the chambers of the tank by the supplier, oralternatively may be dispensed into the chambers by the user. The testsample is placed at predetermined positions on the support media; Thesupport will rest in electrical contact with the semi-solid buffer inthe wells. To start electrophoresis direct current is applied throughthe components of the system. This is accomplished by connecting the byreferring to the following description and claims,

taken in conjunction with the accompanying drawings.

FIG. 1 is a plan view of the electrophoresis unit of the presentinvention.

FIG. 2 is an enlarged plan view of theapparatus of FIG. 1 with the lidremoved.

FIG. 3 is a horizontal sectional view taken along lines 3-3 of FIG. 1.

FIG. 4 is a typical partial sectional view.

FIG. 5 is a partial sectional view identical to FIG. 4 showing a foilelectrode in the operable position.

FIG. 6 is a horizontal sectional view, similar to FIG.

3, in perspective, illustrating a modified form of electrodes.

Referring now particularly to the drawings wherein like referencecharacters indicate like parts, the electrophoresis apparatus shown inFIG. 1 is generally indicated by letter A and includes a substantiallyrectangular electrophoresis chamber 1 having sidewalls 2 and endwalls 3surrounding are extending upwardly from base portions 4 (see FIG. 3).The chamber in FIG. 3 is shown with a lid 5 resting on the top portionthereof. The top portion of the walls 2 and 3 thus provide complementaryengageable surfaces.

FIG. 2, an enlarged view of the unit A of FIG. 1, shows the chamber 1with lid 5 removed. Positioned within the chamber 1 are a pair of buffervessels 6 and 7. The vessels 6 and 7 are open at the top and positionedparallel to the end walls 2 of the chamber 1. Buffer in semi-solid formis placed within the vessels 6 and 7. It is understood that the buffermay be provided by the manufacturer with the apparatus or may be addedby the user. A foil electrode 8 of suitable conducting properties isprovided for retention within each of the buffer vessels 6 and 7 and isshaped to substantially fit within the vessels 6 and 7 as seen in FIG.2. Each foil electrode has an outwardly extending tab portion 9 which isadapted to protrude from the chamber 1. The tabs are connected to asuitable source of direct current 10 through switch 11. The tabs 9 areillustrated in their extended outfolded operable position in FIG. 2.

In FIG. 3 a lid 5 is illustrated in position upon the chamber 1 toeffectively enclose the chamber to air to avoid substantial evaporationtherefrom. A sheet 12 positioned across the top of the two wells 6 and 7has an intermediate support portion adapted to receive and hold asuitable medium for electrophoresis. The medium is in electrical contactwith the semi-solid buffer in buffer vessels 6 and 7.

FIG. 4 shows, in an enlarged fragmentary manner, a portion of the lid 5in place upon the chamber 1 and shows the foil electrode folded backover the semi-solid solvent 7 in the well. The foil electrodes on eitherside is folded back when not in use or for storage purposes.

FIG. 5 is a view similar to FIG. 4 and shows the foil electrode 8 in itsfolded out operable position whereby it resides between thecomplementary engaging wall surfaces of the lid 5 and chamber 1.

FIG. 6 illustrates a modified form of electrodes in which a pair ofwedge shaped knife edge electrodes 13 and 14 are held in holes 14 and 15in the sheet 12 and pierce the respective buffer vessels 6 and 7. Leads16 and 17 extend outwardly of the apparatus from the respectiveelectrodes 13 and 14 and are provided with plug means 18 and 19 forconnection to a power supply as illustrated in FIG. 3.

The above-described electrophoresis unit can, according to thisinvention, be used to separate and/or identify ingredients of numerousmixtures. Examples include the separation of serum proteins and lipoproteins, and in the separation of hemoglobins, the various hem oglobinsfrom which diagnosis can be made for several anemias. Amino acids,sugars and other small molecules can also be separated by this method.Many enzymes besides lactate dehydrogenase are also examined by usingthis equipment. Lactic acid dehydrogenase isoenzymes aid in thediagnosis of myocardial infections and liver disease.

Various modifications and variations of the present invention may bemade without departing from the spirit of the discovery or the scope ofthe appended claims.

What is claimed is:

l. A disposable electrophoresis test unit comprising,

a tank having integrally formed means defining a pair of spaced wellsfor holding a buffer in semi-solid form therein;

a buffer in substantially irreversible semi-solid form placed in each ofsaid spaced wells;

a support media positioned at the top of the tank and in electricalcontact with said semi-solid buffer in the wells; and

means including electrodes within said wells for making electricalcontact with said buffer, and connected to a suitable source of directcurrent to provide passage of current through said buffer and supportmedia in order to move particles according to their electrical chargeand permit charged particle migration for particle group identificationthereof.

2. The electrophoresis test unit of claim 1 including a closuretherefor.

3. The electrophoresis test unit of claim 2 wherein said closure is awrapper of plastic or metal foil.

4. The electrophoresis test unit of claim 2 wherein the top of said tankis rectangular in shape and said closure is a lid hinged to said tankalong an edge thereof, and a raised rib along the remaining edges ofsaid tank is adapted frictionally to engage said lid along its unhingededges to close said tank to the atmosphere.

5. The electrophoresis test unit of claim 1 wherein said semi-solidbuffer is a suspension of a buffer solution in particles of a thickeningagent forming a semisolid substantially irreversible by heat.

6. The electrophoresis unit of claim 5 in which said thickening agent isfumed silica.

7. The electrophoresis test unit of claim 1 wherein said support mediais a thin layer plate.

8. The electrophoresis test unit of claim 1 wherein said chamber is madeof plastic,

The process of separating materials by electrophoresis using theelectrophoresis unit of claim 1 which comprises depositing a test sampleon said buffered support media which is in electrical contact with thesemi-solid buffer in the wells of said unit, and applying direct currentthrough the components of the system for a predetermined length of timeto cause separation of the components of said materials by differentialmigration in the electric field.

10. The process of claim 9 wherein said components which are separatedand identified by analytical means.

1. A disposable electrophoresis test unit comprising, a tank havingintegrally formed means defining a pair of spacEd wells for holding abuffer in semi-solid form therein; a buffer in substantiallyirreversible semi-solid form placed in each of said spaced wells; asupport media positioned at the top of the tank and in electricalcontact with said semi-solid buffer in the wells; and means includingelectrodes within said wells for making electrical contact with saidbuffer, and connected to a suitable source of direct current to providepassage of current through said buffer and support media in order tomove particles according to their electrical charge and permit chargedparticle migration for particle group identification thereof.
 2. Theelectrophoresis test unit of claim 1 including a closure therefor. 3.The electrophoresis test unit of claim 2 wherein said closure is awrapper of plastic or metal foil.
 4. The electrophoresis test unit ofclaim 2 wherein the top of said tank is rectangular in shape and saidclosure is a lid hinged to said tank along an edge thereof, and a raisedrib along the remaining edges of said tank is adapted frictionally toengage said lid along its unhinged edges to close said tank to theatmosphere.
 5. The electrophoresis test unit of claim 1 wherein saidsemi-solid buffer is a suspension of a buffer solution in particles of athickening agent forming a semisolid substantially irreversible by heat.6. The electrophoresis unit of claim 5 in which said thickening agent isfumed silica.
 7. The electrophoresis test unit of claim 1 wherein saidsupport media is a thin layer plate.
 8. The electrophoresis test unit ofclaim 1 wherein said chamber is made of plastic.
 9. The process ofseparating materials by electrophoresis using the electrophoresis unitof claim 1 which comprises depositing a test sample on said bufferedsupport media which is in electrical contact with the semi-solid bufferin the wells of said unit, and applying direct current through thecomponents of the system for a predetermined length of time to causeseparation of the components of said materials by differential migrationin the electric field.